Chemically and metabolically stable dipeptide possessing potent sodium channel blocker activity

ABSTRACT

A very stable, selective and renally safe sodium channel blocker represented by the formula: The invention also includes a variety of compositions, combinations and methods of treatment using this inventive sodium channel blocker.

The present application claims priority to U.S. Ser. No. 61/501,524,filed Jun. 27, 2011, the entire contents of which are herebyincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to epithelial sodium channel blocker3,5-diamino-6-chloro-N—(N-(4-(4-((S)-3-(dimethylamino)-4-((S)-1-(dimethylamino)-6-guanidino-1-oxohexan-2-ylamino)-4-oxobutyl)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide(I). The present invention also includes a variety of methods oftreatment using this inventive sodium channel blocker. The presentinvention also relates to novel compounds for the treatment of dry eye,particularly including3,5-diamino-6-chloro-N—(N-(4-(4-((S)-3-(dimethylamino)-4-((S)-1-(dimethylamino)-6-guanidino-1-oxohexan-2-ylamino)-4-oxobutyl)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide(I) and its pharmaceutically acceptable salt forms, useful as sodiumchannel blockers, compositions containing the same, therapeutic methodsand uses for the same and processes for preparing the same.

2. Description of the Background

The mucosal surfaces at the interface between the environment and thebody have evolved a number of “innate defense”, i.e., protectivemechanisms. A principal form of such innate defense is to cleanse thesesurfaces with liquid. Typically, the quantity of the liquid layer on amucosal surface reflects the balance between epithelial liquidsecretion, often reflecting anion (Cl⁻ and/or HCO₃ ⁻) secretion coupledwith water (and a cation counter-ion), and epithelial liquid absorption,often reflecting Na⁺ absorption, coupled with water and counter anion(Cl⁻ and/or HCO₃ ⁻). Many diseases of mucosal surfaces are caused by toolittle protective liquid on those mucosal surfaces created by animbalance between secretion (too little) and absorption (relatively toomuch). The defective salt transport processes that characterize thesemucosal dysfunctions reside in the epithelial layer of the mucosalsurface.

One approach to replenish the protective liquid layer on mucosalsurfaces is to “re-balance” the system by blocking Na⁺ channel andliquid absorption. The epithelial protein that mediates therate-limiting step of Na⁺ and liquid absorption is the epithelial Na⁺channel (ENaC). ENaC is positioned on the apical surface of theepithelium, i.e. the mucosal surface-environmental interface. Therefore,to inhibit ENaC mediated Na⁺ and liquid absorption, an ENaC blocker ofthe amiloride class (which blocks from the extracellular domain of ENaC)must be delivered to the mucosal surface and, importantly, be maintainedat this site, to achieve therapeutic utility. The present inventiondescribes diseases characterized by too little liquid on mucosalsurfaces and “topical” sodium channel blockers designed to exhibit theincreased potency, reduced mucosal absorption, and slow dissociation(“unbinding” or detachment) from ENaC required for therapy of thesediseases.

Chronic obstructive pulmonary diseases are characterized by dehydrationof airway surfaces and the retention of mucous secretions in the lungs.Examples of such diseases include cystic fibrosis, chronic bronchitis,and primary or secondary ciliary dyskinesia. Such diseases affectapproximately 15 million patients in the United States, and are thesixth leading cause of death. Other airway or pulmonary diseasescharacterized by the accumulation of retained mucous secretions includesinusitis (an inflammation of the paranasal sinuses associated withupper respiratory infection) and pneumonia.

Chronic bronchitis (CB), including the most common lethal genetic formof chronic bronchitis, cystic fibrosis (CF), are diseases that reflectthe body's failure to clear mucus normally from the lungs, whichultimately produces chronic airways infection. In the normal lung, theprimary defense against chronic intrapulmonary airways infection(chronic bronchitis) is mediated by the continuous clearance of mucusfrom bronchial airway surfaces. This function in health effectivelyremoves from the lung potentially noxious toxins and pathogens. Recentdata indicate that the initiating problem, i.e., the “basic defect,” inboth CB and CF is the failure to clear mucus from airway surfaces. Thefailure to clear mucus reflects an imbalance between the amount ofliquid and mucin on airway surfaces. This “airway surface liquid” (ASL)is primarily composed of salt and water in proportions similar to plasma(i.e., isotonic). Mucin macromolecules organize into a well defined“mucus layer” which normally traps inhaled bacteria and is transportedout of the lung via the actions of cilia which beat in a watery, lowviscosity solution termed the “periciliary liquid” (PCL). In the diseasestate, there is an imbalance in the quantities of mucus as ASL on airwaysurfaces. This results in a relative reduction in ASL which leads tomucus concentration, reduction in the lubricant activity of the PCL, anda failure to clear mucus via ciliary activity to the mouth. Thereduction in mechanical clearance of mucus from the lung leads tochronic bacterial colonization of mucus adherent to airway surfaces. Itis the chronic retention of bacteria, the failure of local antimicrobialsubstances to kill mucus-entrapped bacteria on a chronic basis, and theconsequent chronic inflammatory responses of the body to this type ofsurface infection, that lead to the syndromes of CB and CF.

The current afflicted population in the U.S. is 12,000,000 patients withthe acquired (primarily from cigarette smoke exposure) form of chronicbronchitis and approximately 30,000 patients with the genetic form,cystic fibrosis. Approximately equal numbers of both populations arepresent in Europe. In Asia, there is little CF but the incidence of CBis high and, like the rest of the world, is increasing.

There is currently a large, unmet medical need for products thatspecifically treat CB and CF at the level of the basic defect that causethese diseases. The current therapies for chronic bronchitis and cysticfibrosis focus on treating the symptoms and/or the late effects of thesediseases. Thus, for chronic bronchitis, β-agonists, inhaled steroids,anti-cholinergic agents, and oral theophyllines and phosphodiesteraseinhibitors are all in development. However, none of these drugs treateffectively the fundamental problem of the failure to clear mucus fromthe lung. Similarly, in cystic fibrosis, the same spectrum ofpharmacologic agents is used. These strategies have been complemented bymore recent strategies designed to clear the CF lung of the DNA(“Pulmozyme”; Genentech) that has been deposited in the lung byneutrophils that have futilely attempted to kill the bacteria that growin adherent mucus masses and through the use of inhaled antibiotics(“TOBI”) designed to augment the lungs' own killing mechanisms to ridthe adherent mucus plaques of bacteria. A general principle of the bodyis that if the initiating lesion is not treated, in this case mucusretention/obstruction, bacterial infections became chronic andincreasingly refractory to antimicrobial therapy. Thus, a major unmettherapeutic need for both CB and CF lung diseases is an effective meansof re-hydrating airway mucus (i.e., restoring/expanding the volume ofthe ASL) and promoting its clearance, with bacteria, from the lung.

R. C. Boucher, in U.S. Pat. No. 6,264,975, describes the use ofpyrazinoylguanidine sodium channel blockers for hydrating mucosalsurfaces. These compounds, typified by the well-known diureticsamiloride, benzamil, and phenamil, are effective. However, thesecompounds suffer from the significant disadvantage that they are (1)relatively impotent, which is important because the mass of drug thatcan be inhaled by the lung is limited; (2) rapidly absorbed, whichlimits the half-life of the drug on the mucosal surface; and (3) arefreely dissociable from ENaC. The sum of these disadvantages embodied inthese well known diurectics produces compounds with insufficient potencyand/or effective half-life on mucosal surfaces to have therapeuticbenefit for hydrating mucosal surfaces.

R. C. Boucher, in U.S. Pat. No. 6,926,911, suggests the use of therelatively impotent sodium channel blockers such as amiloride, withosmolytes for the treatment of airway diseases. This combination givesno practical advantage over either treatment alone and is clinically notuseful, see Donaldson et al, N Eng J Med., 2006; 353:241-250. Amiloridewas found to block the water permeability of airways and negate thepotential benefit of concurrent use of hypertonic saline and amiloride.

U.S. Pat. No. 5,817,028 to Anderson describes a method for theprovocation of air passage narrowing (for evaluating susceptibility toasthma) and/or the induction of sputum in subjects via the inhalation ofmannitol. It is suggested that the same technique can be used to inducesputum and promote mucociliary clearance. Substances suggested includesodium chloride, potassium chloride, mannitol and dextrose.

Clearly, what is needed are drugs that are more effective at restoringthe clearance of mucus from the lungs of patients with CB/CF. The valueof these new therapies will be reflected in improvements in the qualityand duration of life for both the CF and the CB populations.

Other mucosal surfaces in and on the body exhibit subtle differences inthe normal physiology of the protective surface liquids on theirsurfaces but the pathophysiology of disease reflects a common theme,i.e., too little protective surface liquid. For example, in xerostomia(dry mouth) the oral cavity is depleted of liquid due to a failure ofthe parotid sublingual and submandibular glands to secrete liquiddespite continued Na⁺ (ENaC) transport mediated liquid absorption fromthe oral cavity.

Similarly, keratoconjunctivitis sira (dry eye) is caused by failure oflacrimal glands to secrete liquid in the face of continued Na⁺ dependentliquid absorption on conjunctional surfaces. Keratoconjunctivitis sicca(KCS) or chronic dry eye disease (DED) is one of the most frequentlydiagnosed ocular diseases, resulting in painful irritation, inflammationon the ocular surface, and impaired vision. KCS/DED results frominadequate aqueous tear fluid on the eyes. Dry eye is one of the mostfrequently diagnosed ocular diseases affecting more than 5 millionpeople in the United States alone. Dry eye is a multi-factorial disease,resulting from a common etiology of insufficient tear film causingocular surface damage and symptoms of ocular discomfort. The few currenttherapies available, which include immunosuppressive agents andover-the-counter tear replacements, are not sufficiently efficacious formany users or only provide transient relief from dry eye symptoms.Therefore, the development of novel agents to treat dry eye would be oftremendous benefit to the therapeutic milieu. The volume of tear film onthe ocular surface represents a balance between tear fluid output versusfluid loss via drainage, evaporation, or epithelial absorption. Similarto other epithelial tissues, the epithelium of the conjunctiva andcornea are capable of regulating the hydration status of the mucosalsurface through active salt and water transport. The epithelial sodiumchannel (ENaC) is a key regulator of sodium (and water) absorption innumerous tissues including the eye. The inhibition of ENaC in the eye ispredicted to preserve lacrimal secretions and maintain hydration on theocular surface.

In rhinosinusitis, there is an imbalance, as in CB, between mucinsecretion and relative ASL depletion. Finally, in the gastrointestinaltract, failure to secrete Cl− (and liquid) in the proximal smallintestine, combined with increased Na⁺ (and liquid) absorption in theterminal ileum leads to the distal intestinal obstruction syndrome(DIOS). In older patients excessive Na⁺ (and volume) absorption in thedescending colon produces constipation and diverticulitis.

The published literature includes a number of patent applications andgranted patents to Parion Sciences Inc., directed towardpyrazinoylguanidine analogs as sodium channel blockers. Examples of suchpublications include PCT Publication Nos. WO2003/070182, WO2003/070184,WO2004/073629, WO2005/025496, WO2005/016879, WO2005/018644,WO2006/022935, WO2006/023573, WO2006/023617, WO2007/018640,WO2007/146869, WO2008/031028, WO2008/031048, and U.S. Pat. Nos.6,858,614, 6,858,615, 6,903,105, 7,064,129, 7,186,833, 7,189,719,7,192,958, 7,192,959, 7,192,960, 7,241,766, 7,247,636, 7,247,637,7,317,013, 7,332,496, 7,368,447, 7,368,450, 7,368,451, 7,375,102,7,388,013, 7,399,766, 7,410,968, 7,807,834, 7,842,697, and 7,868,010.

There remains a need for novel sodium channel blocking compounds withenhanced potency and effectiveness on mucosal tissues. There alsoremains the need for novel sodium channel blocking compounds thatprovide therapeutic effect, but minimize or eliminate the onset orprogression of hyperkalemia in recipients.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a compound that isstable in liquid formulations suitable for topical administration.

It is an object of the present invention to provide a compound that ismore potent in vivo and/or absorbed less rapidly from mucosal surfaces,and/or are less reversible as compared to known compounds such asamiloride, benzamil and phenamil.

It is another object of the present invention to provide compounds whichare (1) absorbed less rapidly from mucosal surfaces, especially airwaysurfaces, as compared to known compounds and; (2) when absorbed frommusosal surfaces after administration to the mucosal surfaces, arelargely non renally absorbed so as to prevent renal side effects such ashyperkalemia.

It is another object of the present invention to provide methods oftreatment that take advantage of the pharmacological properties of thecompounds described above.

In particular, it is an object of the present invention to providemethods of treatment which rely on rehydration of mucosal surfaces.

In particular, it is an object of the present invention to providemethods of treating dry eye and related ocular diseases.

The objects of the present invention may be accomplished with apyrazinoylguanidine represented by the compound of formula (I):

The present invention also provides pharmaceutical compositions whichcomprise the compound described herein.

The present invention also provides a method of promoting hydration ofmucosal surfaces, comprising:

administering an effective amount of compound I described herein to amucosal surface of a subject.

The present invention also provides a method of restoring mucosaldefense, comprising:

topically administering an effective amount compound I described hereinto a mucosal surface of a subject in need thereof.

The present invention also provides a method of blocking ENaC,comprising:

contacting sodium channels with an effective amount of compound Irepresented by described herein.

The present invention also provides a method of promoting mucusclearance in mucosal surfaces, comprising:

administering an effective amount of compound I described herein to amucosal surface of a subject.

The present invention also provides a method of treating chronicbronchitis, comprising:

administering an effective amount of compound I described herein to asubject in need thereof.

The present invention also provides a method of treating cysticfibrosis, comprising:

administering an effective amount of compound I described herein to asubject in need thereof.

The present invention also provides a method of treating rhinosinusitis,comprising:

administering an effective amount of compound I described herein to asubject in need thereof.

The present invention also provides a method of treating nasaldehydration, comprising:

administering an effective amount of compound I described herein to thenasal passages of a subject in need thereof.

In a specific embodiment, the nasal dehydration is brought on byadministering dry oxygen to the subject.

The present invention also provides a method of treating sinusitis,comprising:

administering an effective amount of compound I described herein to asubject in need thereof.

The present invention also provides a method of treating pneumonia,comprising:

administering an effective amount of compound I described herein to asubject in need thereof.

The present invention also provides a method of treatingventilator-induced pneumonia, comprising:

administering an effective amount of compound I described herein to asubject by means of a ventilator.

The present invention also provides a method of treating asthma,comprising:

administering an effective amount of compound I described herein to asubject in need thereof.

The present invention also provides a method of treating primary ciliarydyskinesia, comprising:

administering an effective amount of compound I described herein to asubject in need thereof.

The present invention also provides a method of treating otitis media,comprising:

administering an effective amount of compound I described herein to asubject in need thereof.

The present invention also provides a method of inducing sputum fordiagnostic purposes, comprising:

administering an effective amount of compound I described herein to asubject in need thereof.

The present invention also provides a method of treating chronicobstructive pulmonary disease, comprising:

administering an effective amount of compound I described herein to asubject in need thereof.

The present invention also provides a method of treating emphysema,comprising:

administering an effective amount of compound I described herein to asubject in need thereof.

The present invention also provides a method of treating dry eye,comprising:

administering an effective amount of compound I described herein to theeye of the subject in need thereof.

The present invention also provides a method of promoting ocularhydration, comprising:

administering an effective amount of compound I described herein to theeye of the subject.

The present invention also provides a method of promoting cornealhydration, comprising:

administering an effective amount of compound I described herein to theeye of the subject.

The present invention also provides a method of treating Sjögren'sdisease, comprising:

administering an effective amount of compound I described herein to asubject in need thereof.

The present invention also provides a method of treating vaginaldryness, comprising:

administering an effective amount of compound I described herein to thevaginal tract of a subject in need thereof.

The present invention also provides a method of treating dry skin,comprising:

administering an effective amount of compound I described herein to theskin of a subject in need thereof.

The present invention also provides a method of treating dry mouth(xerostomia), comprising:

administering an effective amount of compound I described herein to themouth of the subject in need thereof.

The present invention also provides a method of treating distalintestinal obstruction syndrome, comprising:

administering an effective amount of compound I described herein to asubject in need thereof.

The present invention also provides a method of treating esophagitis,comprising:

administering an effective amount of compound I described herein to asubject in need thereof.

The present invention also provides a method of treating bronchiectasis,comprising:

administering an effective amount of compound I described herein to asubject in need thereof.

The present invention also provides a method of treating constipation,comprising:

administering an effective amount of compound I described herein to asubject in need thereof. In one embodiment of this method, the compoundis administered either orally or via a suppository or enema.

The present invention also provides a method of treating chronicdiverticulitis comprising:

administering an effective amount of compound I described herein to asubject in need thereof.

It is another aspect of the present invention to provide treatmentsusing the sodium channel blocker when administered with an osmoticenhancer. Therefore, such a sodium channel blocker of Formula I whenused in conjunction with osmolytes will give a prolonged pharmacodynamichalf-life on mucosal surfaces as compared to either compound used alone.

It is another object of the present invention to provide treatmentsusing a sodium channel blocker of formula (I) and osmolytes togetherwhich are absorbed less rapidly from mucosal surfaces, especially airwaysurfaces.

It is another object of the invention to provide compositions whichcontain a sodium channel blocker of formula (I) and osmolytes.

The objects of the invention may be accomplished with a method oftreating a disease ameliorated by increased mucociliary clearance andmucosal hydration comprising administering an effective amount of thecompound of formula (I) as defined herein and an osmolyte to a subjectto a subject in need of increased mucociliary clearance and mucosalhydration.

The objects of the invention may also be accomplished with a method ofinducing sputum for diagnostic purposes, comprising administering aneffective amount of the compound of formula (I) as defined herein and anosmolyte to a subject in need thereof.

The objects of the invention may also be accomplished with a method oftreating anthrax, comprising administering an effective amount of thecompound of formula (I) as defined herein and an osmolyte to a subjectin need thereof.

The objects of the invention may also be accomplished with a method ofprophylactic, post-exposure prophylactic, preventive or therapeutictreatment against diseases or conditions caused by pathogens,particularly pathogens which may be used in bioterrorism, comprisingadministering an effective amount of the compound of formula (I) to asubject in need thereof.

The objects of the invention may also be accomplished with acomposition, comprising the compound of formula (I) as defined hereinand an osmotically active compound.

DESCRIPTION OF THE FIGURES

FIG. 1: Dose Response Curve of ENaC Inhibition with P-1046 (I) andAmiloride. The potency of P-1046 compared to amiloride was generated inUssing chamber studies using primary cultures of canine bronchialepithelial cells. P-1046 is >100-fold more potent than amiloride asindicated by the leftward shift in the dose response curve.

FIG. 2: The Effects of P-1046 (Formula I, 15) Concentration on TearOutput in ExLac Rats. The raw PRT tear output values are shown in theleft panel and the baseline corrected values are shown in the rightpanel. At all concentrations, P-1046 increases tear output in ExLac rats15 minutes post-dose at or slightly below tear output values observedfor normal rats. The effects are still observed 2 hours post-dose, withthe exception of the 0.1 mM dose group. n=4 for all groups.

FIG. 3: The Clearance of P-1046 (Formula I, 15) from the Ocular Surface.The concentration of P-1046 was determined using the proceduresdescribed above by determining the mass of P-1046 extracted from eachthread and determining the concentration based on the volume of fluidwicked onto the thread. For all concentrations tested, P-1046 exhibitsan apparent bi-phasic clearance, whereby the majority of the drug iscleared from the ocular surface by 30 minutes post-dose, followed by aslow clearance phase from 30 to 120 minutes post-dose. Note, theconcentration of P-1046 during the slow clearance phase forconcentration ≧1 mM are well above the IC50 for P-1046 (5.3 nM), therebyproviding the long lasting increase in tear volume.

FIG. 4: FIG. 4. The Effects of 10 mM P-1046 (Formula I, 15) on TearOutput in ExLac Rats. The raw PRT tear output values are shown in theleft panel and the baseline corrected values are shown in the rightpanel. A single 10 mM dose of P-1046 produces increased tear volumerelative to vehicle controls. While the raw tear output data are notsignificant beyond 60 minutes (reflecting the higher baseline values forcontrol animals relative to P-1046 treated animals), the baselinecorrected data are statistically significant at all post-dose timepoints (p<0.03) n=3 for both groups.

FIG. 5: HPLC Analysis of P-1046 (Formula I, 15) solubility/stabilitysamples at Day 1 and Day 10. No degradation of P-1046 was observed after10 days at 50° C. in pH 4.2 citrate buffer with 2.8% NaCl. P-1046 wasfully soluble in this buffer at the highest concentration tested 8.8mg/mL.

FIG. 6: Summary of P-1046 Ocular Tolerability. (A) The cumulative Draizescores for each dose group and each dosing interval (the data are thesum of all Draize scores for the right eye). Both dosing regimens forP-1046 show only slight increases in Draize scoring which are notgreater than those observed for vehicle treated animals. For comparison,a compound identified as irritating in the same study is shown inyellow. (B) The average blink rate for each dose group at each dosinginterval. Both dosing regimens for P-1046 show only slight increases atsome time points, similarly to vehicle treated animals. These datasuggest that P-1046 at 10 and 30 mM do not cause stinging oninstallation.

FIG. 7: P-1046 Plasma Levels after Ocular Dosing. (A) The plasma levelsfrom individual animals during and after 8 doses of 10 mM P-1046 (50ul/dose). (B) The plasma levels from individual animals during and after4 doses of 30 mM P-1046 (50 ul/dose).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that the compound offormula (I) is more potent and/or absorbed less rapidly from mucosalsurfaces, especially airway surfaces, compared to known sodium channelblockers such as amilorde, benzamil, and phenamil. Therefore, thecompound of formula (I) has a longer half-life on mucosal surfaces ascompared to these compounds.

The present invention is also based on the discovery that certaincompounds embraced by formula (I) are (1) absorbed less rapidly frommucosal surfaces, especially ocular surfaces, as compared to knowncompounds and (2) when absorbed from musosal surfaces afteradministration to the mucosal surfaces, are excreted mainly non-renallyin order to minimize the chances of hyperkalemia.

The present invention is also based on the discovery that certaincompounds embraced by formula (I) provide methods of treatment that takeadvantage of the pharmacological properties of the compounds describedabove.

In particular, the present invention is also based on the discovery thatcertain compounds embraced by formula (I) rehydrate of mucosal surfaces.

In particular, the present invention is also based on the discovery thatcertain compounds embraced by formula (I) are useful in treating dry eyeand related ocular diseases.

Compound I described herein may be prepared and used as the free base.Alternatively, the compound may be prepared and used as apharmaceutically acceptable salt. Pharmaceutically acceptable salts aresalts that retain or enhance the desired biological activity of theparent compound and do not impart undesired toxicological effects.Examples of such salts are (a) acid addition salts formed with inorganicacids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid, nitric acid and the like; (b) salts formed with organicacids such as, for example, acetic acid, oxalic acid, tartaric acid,succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid,malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid,alginic acid, polyglutamic acid, naphthalenesulfonic acid,methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonicacid, polygalacturonic acid, malonic acid, sulfosalicylic acid, glycolicacid, 2-hydroxy-3-naphthoate, pamoate, salicylic acid, stearic acid,phthalic acid, mandelic acid, lactic acid and the like; and (c) saltsformed from elemental anions for example, chlorine, bromine, and iodine.

It is to be noted that all enantiomers, diastereomers, and racemicmixtures, tautomers, polymorphs, pseudopolymorphs and pharmaceuticallyacceptable salts of the compound within the scope of formula (I) areembraced by the present invention. All mixtures of such enantiomers anddiastereomers are within the scope of the present invention.

A compound of formula I and its pharmaceutically acceptable salts mayexist as different polymorphs or pseudopolymorphs. As used herein,crystalline polymorphism means the ability of a crystalline compound toexist in different crystal structures. The crystalline polymorphism mayresult from differences in crystal packing (packing polymorphism) ordifferences in packing between different conformers of the same molecule(conformational polymorphism). As used herein, crystallinepseudopolymorphism means the ability of a hydrate or solvate of acompound to exist in different crystal structures. The pseudopolymorphsof the instant invention may exist due to differences in crystal packing(packing pseudopolymorphism) or due to differences in packing betweendifferent conformers of the same molecule (conformationalpseudopolymorphism). The instant invention comprises all polymorphs andpseudopolymorphs of the compounds of formula I and theirpharmaceutically acceptable salts.

A compound of formula I and its pharmaceutically acceptable salts mayalso exist as an amorphous solid. As used herein, an amorphous solid isa solid in which there is no long-range order of the positions of theatoms in the solid. This definition applies as well when the crystalsize is two nanometers or less. Additives, including solvents, may beused to create the amorphous forms of the instant invention. The instantinvention comprises all amorphous forms of the compounds of formulaI-III and their pharmaceutically acceptable salts.

The compound of formula I may exist in different tautomeric forms. Oneskilled in the art will recognize that guanidines can exist intautomeric forms. By way of example and not by way of limitation,compounds of formula I can exist in various tautomeric forms as shownbelow:

All possible tautomeric forms of the guanidines and acyl guanidines allof the embodiments of formula I are within the scope of the instantinvention.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., NewYork. Many organic compounds exist in optically active forms, i.e., theyhave the ability to rotate the plane of plane-polarized light. Indescribing an optically active compound, the prefixes D and L or R and Sare used to denote the absolute configuration of the molecule about itschiral center(s). The prefixes d and 1, D and L, or (+) and (−) areemployed to designate the sign of rotation of plane-polarized light bythe compound, with S, (−), or 1 meaning that the compound islevorotatory while a compound prefixed with R, (+), or d isdextrorotatory. For a given chemical structure, these stereoisomers areidentical except that they are mirror images of one another. A specificstereoisomer may also be referred to as an enantiomer, and a mixture ofsuch isomers is often called an enantiomeric mixture. A 50:50 mixture ofenantiomers is referred to as a racemic mixture or a racemate, which mayoccur where there has been no stereoselection or stereospecificity in achemical reaction or process. The terms “racemic mixture” and “racemate”refer to an equimolar mixture of two enantiomeric species, devoid ofoptical activity.

A single stereoisomer, e.g. an enantiomer, substantially free of itsstereoisomer may be obtained by resolution of the racemic mixture usinga method such as formation of diastereomers using optically activeresolving agents (“Stereochemistry of Carbon Compounds,” (1962) by E. L.Eliel, McGraw Hill; Lochmuller, C. H., (1975) J. Chromatogr., 113:(3)283-302). Racemic mixtures of chiral compounds of the invention can beseparated and isolated by any suitable method, including: (1) formationof ionic, diastereomeric salts with chiral compounds and separation byfractional crystallization or other methods, (2) formation ofdiastereomeric compounds with chiral derivatizing reagents, separationof the diastereomers, and conversion to the pure stereoisomers, and (3)separation of the substantially pure or enriched stereoisomers directlyunder chiral conditions.

The term “diastereomer” refers to a stereoisomer with two or morecenters of chirality and whose molecules are not mirror images of oneanother. Diastereomers have different physical properties, e.g. meltingpoints, boiling points, spectral properties, and reactivities. Mixturesof diastereomers may separate under high resolution analyticalprocedures such as electrophoresis and chromatography.

Without being limited to any particular theory, it is believed that thecompound of formula (I) in vivo as sodium channel blockers. By blockingepithelial sodium channels present in mucosal surfaces the compounds offormula (I) reduce the absorption of water by the mucosal surfaces. Thiseffect increases the volume of protective liquids on mucosal surfaces,rebalances the system, and thus treats disease.

The present invention also provides methods of treatment that takeadvantage of the properties of the compounds described herein asdiscussed above. Thus, subjects that may be treated by the methods ofthe present invention include, but are not limited to, patientsafflicted with cystic fibrosis, primary ciliary dyskinesia, chronicbronchitis, bronchiectasis chronic obstructive airway disease,artificially ventilated patients, patients with acute pneumonia, etc.The present invention may be used to obtain a sputum sample from apatient by administering the active compounds to at least one lung of apatient, and then inducing or collecting a sputum sample from thatpatient. Typically, the invention will be administered to respiratorymucosal surfaces via aerosol (liquid or dry powders) or lavage.

Subjects that may be treated by the method of the present invention alsoinclude patients being administered supplemental oxygen nasally (aregimen that tends to dry the airway surfaces); patients afflicted withan allergic disease or response (e.g., an allergic response to pollen,dust, animal hair or particles, insects or insect particles, etc.) thataffects nasal airway surfaces; patients afflicted with a bacterialinfection e.g., staphylococcus infections such as Staphylococcus aureusinfections, Hemophilus influenza infections, Streptococcus pneumoniaeinfections, Pseudomonas aeuriginosa infections, etc.) of the nasalairway surfaces; patients afflicted with an inflammatory disease thataffects nasal airway surfaces; or patients afflicted with sinusitis(wherein the active agent or agents are administered to promote drainageof congested mucous secretions in the sinuses by administering an amounteffective to promote drainage of congested fluid in the sinuses), orcombined, Rhinosinusitis. The invention may be administered torhino-sinal surfaces by topical delivery, including aerosols and drops.

The present invention may be used to hydrate mucosal surfaces other thanairway surfaces. Such other mucosal surfaces include gastrointestinalsurfaces, oral surfaces, genito-urethral surfaces, ocular surfaces orsurfaces of the eye, the inner ear and the middle ear. For example, theactive compounds of the present invention may be administered by anysuitable means, including locally/topically, orally, or rectally, in aneffective amount.

The present invention is concerned primarily with the treatment of humansubjects, but may also be employed for the treatment of other mammaliansubjects, such as dogs and cats, for veterinary purposes.

As discussed above, the compounds used to prepare the compositions ofthe present invention may be in the form of a pharmaceuticallyacceptable free base. Because the free base of the compound is generallyless soluble in aqueous solutions than the salt, free base compositionsare employed to provide more sustained release of active agent to thelungs. An active agent present in the lungs in particulate form whichhas not dissolved into solution is not available to induce aphysiological response, but serves as a depot of bioavailable drug whichgradually dissolves into solution.

Another aspect of the present invention is a pharmaceutical composition,comprising a compound of formula (I) in a pharmaceutically acceptablecarrier (e.g., an aqueous carrier solution). In general, the compound offormula (I) is included in the composition in an amount effective toinhibit the reabsorption of water by mucosal surfaces.

Without being limited to any particular theory, it is believed thatsodium channel blockers of the present invention block epithelial sodiumchannels present in mucosal surfaces the sodium channel blocker,described herein reduce the absorption of salt and water by the mucosalsurfaces. This effect increases the volume of protective liquids onmucosal surfaces, rebalances the system, and thus treats disease. Thiseffect is enhanced when used in combination with osmolytes.

The compounds of formula (I) may also be used in conjunction withosmolytes thus lowering the dose of the compound needed to hydratemucosal surfaces. This important property means that the compound willhave a lower tendency to cause undesired side-effects by blocking sodiumchannels located at untargeted locations in the body of the recipient,e.g., in the kidneys when used in combination with an osmolyte.

Active osmolytes of the present invention are molecules or compoundsthat are osmotically active (i.e., are “osmolytes”). “Osmoticallyactive” compounds of the present invention are membrane-impermeable(i.e., essentially non-absorbable) on the airway or pulmonary epithelialsurface. The terms “airway surface” and “pulmonary surface,” as usedherein, include pulmonary airway surfaces such as the bronchi andbronchioles, alveolar surfaces, and nasal and sinus surfaces. Activecompounds of the present invention may be ionic osmolytes (i.e., salts),or may be non-ionic osmolytes (i.e., sugars, sugar alcohols, and organicosmolytes). It is specifically intended that both racemic forms of theactive compounds that are racemic in nature are included in the group ofactive compounds that are useful in the present invention. It is to benoted that all racemates, enantiomers, diastereomers, tautomers,polymorphs and pseudopolymorphs and racemic mixtures of the osmoticallyactive compounds are embraced by the present invention.

Active osmolytes useful in the present invention that are ionicosmolytes include any salt of a pharmaceutically acceptable anion and apharmaceutically acceptable cation. Preferably, either (or both) of theanion and cation are non-absorbable (i.e., osmotically active and notsubject to rapid active transport) in relation to the airway surfaces towhich they are administered. Such compounds include but are not limitedto anions and cations that are contained in FDA approved commerciallymarketed salts, see, e.g., Remington: The Science and Practice ofPharmacy, Vol. II, pg. 1457 (19^(th) Ed. 1995), incorporated herein byreference, and can be used in any combination including theirconventional combinations.

Pharmaceutically acceptable osmotically active anions that can be usedto carry out the present invention include, but are not limited to,acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide,calcium edetate, camsylate (camphorsulfonate), carbonate, chloride,citrate, dihydrochloride, edetate, edisylate (1,2-ethanedisulfonate),estolate (lauryl sulfate), esylate (1,2-ethanedisulfonate), fumarate,gluceptate, gluconate, glutamate, glycollylarsanilate(p-glycollamidophenylarsonate), hexylresorcinate, hydrabamine(N,N′-Di(dehydroabietyl)ethylenediamine), hydrobromide, hydrochloride,hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate,maleate, mandelate, mesylate, methylbromide, methylnitrate,methylsulfate, mucate, napsylate, nitrate, nitrte, pamoate (embonate),pantothenate, phosphate or diphosphate, polygalacturonate, salicylate,stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate(8-chlorotheophyllinate), triethiodide, bicarbonate, etc. Particularlypreferred anions include chloride, sulfate, nitrate, gluconate, iodide,bicarbonate, bromide, and phosphate.

Pharmaceutically acceptable cations that can be used to carry out thepresent invention include, but are not limited to, organic cations suchas benzathine (N,N′-dibenzylethylenediamine), chloroprocaine, choline,diethanolamine, ethylenediamine, meglumine (N-methyl D-glucamine),procaine, D-lysine, L-lysine, D-arginine, L-arginine, triethylammonium,N-methyl D-glycerol, and the like. Particularly preferred organiccations are 3-carbon, 4-carbon, 5-carbon and 6-carbon organic cations.Metallic cations useful in the practice of the present invention includebut are not limited to aluminum, calcium, lithium, magnesium, potassium,sodium, zinc, iron, ammonium, and the like. Particularly preferredcations include sodium, potassium, choline, lithium, meglumine,D-lysine, ammonium, magnesium, and calcium.

Specific examples of osmotically active salts that may be used with thesodium channel blockers described herein to carry out the presentinvention include, but are not limited to, sodium chloride, potassiumchloride, choline chloride, choline iodide, lithium chloride, megluminechloride, L-lysine chloride, D-lysine chloride, ammonium chloride,potassium sulfate, potassium nitrate, potassium gluconate, potassiumiodide, ferric chloride, ferrous chloride, potassium bromide, etc.Either a single salt or a combination of different osmotically activesalts may be used to carry out the present invention. Combinations ofdifferent salts are preferred. When different salts are used, one of theanion or cation may be the same among the differing salts.

Osmotically active compounds of the present invention also includenon-ionic osmolytes such as sugars, sugar-alcohols, and organicosmolytes. Sugars and sugar-alcohols useful in the practice of thepresent invention include but are not limited to 3-carbon sugars (e.g.,glycerol, dihydroxyacetone); 4-carbon sugars (e.g., both the D and Lforms of erythrose, threose, and erythrulose); 5-carbon sugars (e.g.,both the D and L forms of ribose, arabinose, xylose, lyxose, psicose,fructose, sorbose, and tagatose); and 6-carbon sugars (e.g., both the Dand L forms of altose, allose, glucose, mannose, gulose, idose,galactose, and talose, and the D and L forms of allo-heptulose,allo-hepulose, gluco-heptulose, manno-heptulose, gulo-heptulose,ido-heptulose, galacto-heptulose, talo-heptulose). Additional sugarsuseful in the practice of the present invention include raffinose,raffinose series oligosaccharides, and stachyose. Both the D and L formsof the reduced form of each sugar/sugar alcohol useful in the presentinvention are also active compounds within the scope of the invention.For example, glucose, when reduced, becomes sorbitol; within the scopeof the invention, sorbitol and other reduced forms of sugar/sugaralcohols (e.g., mannitol, dulcitol, arabitol) are accordingly activecompounds of the present invention.

Osmotically active compounds of the present invention additionallyinclude the family of non-ionic osmolytes termed “organic osmolytes.”The term “organic osmolytes” is generally used to refer to moleculesused to control intracellular osmolality in the kidney. See e.g., J. S.Handler et al., Comp. Biochem. Physiol, 117, 301-306 (1997); M. Burg,Am. J. Physiol. 268, F983-F996 (1995), each incorporated herein byreference. Although the inventor does not wish to be bound to anyparticular theory of the invention, it appears that these organicosmolytes are useful in controlling extracellular volume on theairway/pulmonary surface. Organic osmolytes useful as active compoundsin the present invention include but are not limited to three majorclasses of compounds: polyols (polyhydric alcohols), methylamines, andamino acids. The polyol organic osmolytes considered useful in thepractice of this invention include, but are not limited to, inositol,myo-inositol, and sorbitol. The methylamine organic osmolytes useful inthe practice of the invention include, but are not limited to, choline,betaine, carnitine (L-, D- and DL forms), phosphorylcholine,lyso-phosphorylcholine, glycerophosphorylcholine, creatine, and creatinephosphate. The amino acid organic osmolytes of the invention include,but are not limited to, the D- and L-forms of glycine, alanine,glutamine, glutamate, aspartate, proline and taurine. Additionalosmolytes useful in the practice of the invention include tihulose andsarcosine. Mammalian organic osmolytes are preferred, with human organicosmolytes being most preferred. However, certain organic osmolytes areof bacterial, yeast, and marine animal origin, and these compounds arealso useful active compounds within the scope of the present invention.

Under certain circumstances, an osmolyte precursor may be administeredto the subject; accordingly, these compounds are also useful in thepractice of the invention. The term “osmolyte precursor” as used hereinrefers to a compound which is converted into an osmolyte by a metabolicstep, either catabolic or anabolic. The osmolyte precursors of thisinvention include, but are not limited to, glucose, glucose polymers,glycerol, choline, phosphatidylcholine, lyso-phosphatidylcholine andinorganic phosphates, which are precursors of polyols and methylamines.Precursors of amino acid osmolytes within the scope of this inventioninclude proteins, peptides, and polyamino acids, which are hydrolyzed toyield osmolyte amino acids, and metabolic precursors which can beconverted into osmolyte amino acids by a metabolic step such astransamination. For example, a precursor of the amino acid glutamine ispoly-L-glutamine, and a precursor of glutamate is poly-L-glutamic acid.

Also intended within the scope of this invention are chemically modifiedosmolytes or osmolyte precursors. Such chemical modifications involvelinking to the osmolyte (or precursor) an additional chemical groupwhich alters or enhances the effect of the osmolyte or osmolyteprecursor (e.g., inhibits degradation of the osmolyte molecule). Suchchemical modifications have been utilized with drugs or prodrugs and areknown in the art. (See, for example, U.S. Pat. Nos. 4,479,932 and4,540,564; Shek, E. et al., J. Med. Chem. 19:113-117 (1976); Bodor, N.et al., J. Pharm. Sci. 67:1045-1050 (1978); Bodor, N. et al., J. Med.Chem. 26:313-318 (1983); Bodor, N. et al., J. Pharm. Sci. 75:29-35(1986), each incorporated herein by reference.

In general, osmotically active compounds of the present invention (bothionic and non-ionic) that do not promote, or in fact deter or retardbacterial growth are preferred.

The compounds of formula (I) described herein and osmotically activecompounds disclosed herein may be administered in any order and/orconcurrently to mucousal surfaces such as the eye, the nose, and airwaysurfaces including the nasal passages, sinuses and lungs of a subject byany suitable means known in the art, such as by nose drops, mists,aerosols, continuous overnight nasal cannulation, etc. In one embodimentof the invention, the compounds of formula (I) and osmotically activecompounds of the present invention are administered concurrently bytransbronchoscopic lavage. In a preferred embodiment of the invention,the compounds of formula (I) and osmotically active compounds of thepresent invention are deposited on lung airway surfaces by administeringby inhalation an respirable aerosol respirable particles comprised ofthe compounds of formula (I) and the osmotically active compounds, inwhich the compounds of formula (I) can precede or follow the independentdelivery of an osmotically active compound within a sufficiently shorttime for their effects to be additive. The respirable particles may beliquid or solid. Numerous inhalers for administering aerosol particlesto the lungs of a subject are known. In another preferred embodiment ofthe invention, the compounds of formula (I) and osmotically activecompounds can be given concurrently as defined herein.

The compounds of formula (I) and osmotically active compounds of thepresent invention are administered sequentially (in any order) orconcurrently to the subject in need thereof. As used herein, the term“concurrently” means sufficiently close in time to produce a combinedeffect (that is, concurrently may be simultaneously, or it may be two ormore events occurring within a short time period before or after eachother). Concurrently also embraces the delivery of the compounds offormula (I) and osmolytes as a mixture or solution of the two componentsas well as when delivered from two different nebulizers. An example ofthat would be the delivery of compound 1 in one nebulizer and hypertonicsaline in a second nebulizer connected by a T-piece. When administeredwith other active agents, the active compounds of the present inventionmay function as a vehicle or carrier for the other active agent, or maysimply be administered concurrently with the other active agent. Theactive compound of the present invention may be used as a dry or liquidvehicle for administering other active ingredients to airway surfaces.Such other active agents may be administered for treating the disease ordisorder for which they are intended, in their conventional manner anddosages, in combination with the active compounds of the presentinvention, which may be thought of as serving as a vehicle or carrierfor the other active agent. Any such other active ingredient may beemployed, particularly where hydration of the airway surfaces (i.e., theactivity of the osmotically active compounds of the present invention)facilitates the activity of the other active ingredient (e.g., byfacilitating or enhancing uptake of the active ingredient, bycontributing to the mechanism of action of the other active ingredient,or by any other mechanisms). In a preferred embodiment of the invention,when the active compound of the present invention is administeredconcurrently with another active agent, the active compound of thepresent invention has an additive effect in relation to the other activeagent; that is, the desired effect of the other active agent is enhancedby the concurrent administration of the active compounds of the presentinvention.

The compounds of formula (I) of the present invention are also usefulfor treating airborne infections. Examples of airborne infectionsinclude, for example, RSV. The compounds of formula (I) of the presentinvention are also useful for treating an anthrax infection. The presentinvention relates to the use of the compounds of formula (I) of thepresent invention for prophylactic, post-exposure prophylactic,preventive or therapeutic treatment against diseases or conditionscaused by pathogens. In a preferred embodiment, the present inventionrelates to the use of the compounds of formula (I) for prophylactic,post-exposure prophylactic, preventive or therapeutic treatment againstdiseases or conditions caused by pathogens which may be used inbioterrorism.

In recent years, a variety of research programs and biodefense measureshave been put into place to deal with concerns about the use ofbiological agents in acts of terrorism. These measures are intended toaddress concerns regarding bioterrorism or the use of microorganisms orbiological toxins to kill people, spread fear, and disrupt society. Forexample, the National Institute of Allergy and Infectious Diseases(NIAID) has developed a Strategic Plan for Biodefense Research whichoutlines plans for addressing research needs in the broad area ofbioterrorism and emerging and reemerging infectious diseases. Accordingto the plan, the deliberate exposure of the civilian population of theUnited States to Bacillus anthracis spores revealed a gap in thenation's overall preparedness against bioterrorism. Moreover, the reportdetails that these attacks uncovered an unmet need for tests to rapidlydiagnose, vaccines and immunotherapies to prevent, and drugs andbiologics to cure disease caused by agents of bioterrorism.

Much of the focus of the various research efforts has been directed tostudying the biology of the pathogens identified as potentiallydangerous as bioterrorism agents, studying the host response againstsuch agents, developing vaccines against infectious diseases, evaluatingthe therapeutics currently available and under investigation againstsuch agents, and developing diagnostics to identify signs and symptomsof threatening agents. Such efforts are laudable but, given the largenumber of pathogens which have been identified as potentially availablefor bioterrorism, these efforts have not yet been able to providesatisfactory responses for all possible bioterrorism threats.Additionally, many of the pathogens identified as potentially dangerousas agents of bioterrorism do not provide adequate economic incentivesfor the development of therapeutic or preventive measures by industry.Moreover, even if preventive measures such as vaccines were availablefor each pathogen which may be used in bioterrorism, the cost ofadministering all such vaccines to the general population isprohibitive.

Until convenient and effective treatments are available against everybioterrorism threat, there exists a strong need for preventative,prophylactic or therapeutic treatments which can prevent or reduce therisk of infection from pathogenic agents.

The present invention provides such methods of prophylactic treatment.In one aspect, a prophylactic treatment method is provided comprisingadministering a prophylactically effective amount of the compounds offormula (I) to an individual in need of prophylactic treatment againstinfection from one or more airborne pathogens. A particular example ofan airborne pathogen is anthrax.

In another aspect, a prophylactic treatment method is provided forreducing the risk of infection from an airborne pathogen which can causea disease in a human, said method comprising administering an effectiveamount of the compounds of formula (I) to the lungs of the human who maybe at risk of infection from the airborne pathogen but is asymptomaticfor the disease, wherein the effective amount of a sodium channelblocker and osmolye are sufficient to reduce the risk of infection inthe human. A particular example of an airborne pathogen is anthrax.

In another aspect, a post-exposure prophylactic treatment or therapeutictreatment method is provided for treating infection from an airbornepathogen comprising administering an effective amount of the compoundsof formula (I) to the lungs of an individual in need of such treatmentagainst infection from an airborne pathogen. The pathogens which may beprotected against by the prophylactic post exposure, rescue andtherapeutic treatment methods of the invention include any pathogenswhich may enter the body through the mouth, nose or nasal airways, thusproceeding into the lungs. Typically, the pathogens will be airbornepathogens, either naturally occurring or by aerosolization. Thepathogens may be naturally occurring or may have been introduced intothe environment intentionally after aerosolization or other method ofintroducing the pathogens into the environment. Many pathogens which arenot naturally transmitted in the air have been or may be aerosolized foruse in bioterrorism. The pathogens for which the treatment of theinvention may be useful includes, but is not limited to, category A, Band C priority pathogens as set forth by the NIAID. These categoriescorrespond generally to the lists compiled by the Centers for DiseaseControl and Prevention (CDC). As set up by the CDC, Category A agentsare those that can be easily disseminated or transmittedperson-to-person, cause high mortality, with potential for major publichealth impact. Category B agents are next in priority and include thosethat are moderately easy to disseminate and cause moderate morbidity andlow mortality. Category C consists of emerging pathogens that could beengineered for mass dissemination in the future because of theiravailability, ease of production and dissemination and potential forhigh morbidity and mortality. Particular examples of these pathogens areanthrax and plague. Additional pathogens which may be protected againstor the infection risk therefrom reduced include influenza viruses,rhinoviruses, adenoviruses and respiratory syncytial viruses, and thelike. A further pathogen which may be protected against is thecoronavirus which is believed to cause severe acute respiratory syndrome(SARS).

The compounds of the present invention may also be used in conjunctionwith a P2Y2 receptor agonist or a pharmaceutically acceptable saltthereof (also sometimes referred to as an “active agent” herein). Thecomposition may further comprise a P2Y2 receptor agonist or apharmaceutically acceptable salt thereof (also sometimes referred to asan “active agent” herein). The P2Y2 receptor agonist is typicallyincluded in an amount effective to stimulate chloride and watersecretion by airway surfaces, particularly nasal airway surfaces.Suitable P2Y2 receptor agonists are described in columns 9-10 of U.S.Pat. No. 6,264,975, U.S. Pat. No. 5,656,256, and U.S. Pat. No.5,292,498, each of which is incorporated herein by reference.

Bronchodiloators can also be used in combination with compounds of thepresent invention. These bronchodilators include, but are not limitedto, β-adrenergic agonists including but not limited to epinephrine,isoproterenol, fenoterol, albutereol, terbutalin, pirbuterol,bitolterol, metaproterenol, iosetharine, salmeterol xinafoate, as wellas anticholinergic agents including but not limited to ipratropiumbromide, as well as compounds such as theophylline and aminophylline.These compounds may be administered in accordance with known techniques,either prior to or concurrently with the active compounds describedherein.

Another aspect of the present invention is a pharmaceutical formulation,comprising an active compound as described above in a pharmaceuticallyacceptable carrier (e.g., an aqueous carrier solution). In general, theactive compound is included in the composition in an amount effective totreat mucosal surfaces, such as inhibiting the reabsorption of water bymucosal surfaces, including airway and other surfaces.

The active compounds disclosed herein may be administered to mucosalsurfaces by any suitable means, including topically, orally, rectally,vaginally, ocularly and dermally, etc. For example, for the treatment ofconstipation, the active compounds may be administered orally orrectally to the gastrointestinal mucosal surface. The active compoundmay be combined with a pharmaceutically acceptable carrier in anysuitable form, such as sterile physiological or dilute saline or topicalsolution, as a droplet, tablet or the like for oral administration, as asuppository for rectal or genito-urethral administration, etc.Excipients may be included in the formulation to enhance the solubilityof the active compounds, as desired.

The active compounds disclosed herein may be administered to the airwaysurfaces of a patient by any suitable means, including as a spray, mist,or droplets of the active compounds in a pharmaceutically acceptablecarrier such as physiological or dilute saline solutions or distilledwater. For example, the active compounds may be prepared as formulationsand administered as described in U.S. Pat. No. 5,789,391 to Jacobus, thedisclosure of which is incorporated by reference herein in its entirety.

Solid or liquid particulate active agents prepared for practicing thepresent invention could, as noted above, include particles of respirableor non-respirable size; that is, for respirable particles, particles ofa size sufficiently small to pass through the mouth and larynx uponinhalation and into the bronchi and alveoli of the lungs, and fornon-respirable particles, particles sufficiently large to be retained inthe nasal airway passages rather than pass through the larynx and intothe bronchi and alveoli of the lungs. In general, particles ranging fromabout 1 to 5 microns in size (more particularly, less than about 4.7microns in size) are respirable. Particles of non-respirable size aregreater than about 5 microns in size, up to the size of visibledroplets. Thus, for nasal administration, a particle size in the rangeof 10-500 μm may be used to ensure retention in the nasal cavity.

In the manufacture of a formulation according to the invention, activeagents or the physiologically acceptable salts or free bases thereof aretypically admixed with, inter alia, an acceptable carrier. Of course,the carrier must be compatible with any other ingredients in theformulation and must not be deleterious to the patient. The carrier mustbe solid or liquid, or both, and is preferably formulated with thecompound as a unit-dose formulation, for example, a capsule, that maycontain 0.5% to 99% by weight of the active compound. One or more activecompounds may be incorporated in the formulations of the invention,which formulations may be prepared by any of the well-known techniquesof pharmacy consisting essentially of admixing the components.

Compositions containing respirable or non-respirable dry particles ofmicronized active agent may be prepared by grinding the dry active agentwith a mortar and pestle, and then passing the micronized compositionthrough a 400 mesh screen to break up or separate out largeagglomerates.

The particulate active agent composition may optionally contain adispersant which serves to facilitate the formulation of an aerosol. Asuitable dispersant is lactose, which may be blended with the activeagent in any suitable ratio (e.g., a 1 to 1 ratio by weight).

Active compounds disclosed herein may be administered to airway surfacesincluding the nasal passages, sinuses and lungs of a subject by asuitable means know in the art, such as by nose drops, mists, etc. Inone embodiment of the invention, the active compounds of the presentinvention and administered by transbronchoscopic lavage. In a preferredembodiment of the invention, the active compounds of the presentinvention are deposited on lung airway surfaces by administering anaerosol suspension of respirable particles comprised of the activecompound, which the subject inhales. The respirable particles may beliquid or solid. Numerous inhalers for administering aerosol particlesto the lungs of a subject are known.

Inhalers such as those developed by Inhale Therapeutic Systems, PaloAlto, Calif., USA, may be employed, including but not limited to thosedisclosed in U.S. Pat. Nos. 5,740,794; 5,654,007; 5,458,135; 5,775,320;and 5,785,049, each of which is incorporated herein by reference. TheApplicant specifically intends that the disclosures of all patentreferences cited herein be incorporated by reference herein in theirentirety. Inhalers such as those developed by Dura Pharmaceuticals,Inc., San Diego, Calif., USA, may also be employed, including but notlimited to those disclosed in U.S. Pat. Nos. 5,622,166; 5,577,497;5,645,051; and 5,492,112, each of which is incorporated herein byreference. Additionally, inhalers such as those developed by AradigmCorp., Hayward, Calif., USA, may be employed, including but not limitedto those disclosed in U.S. Pat. Nos. 5,826,570; 5,813,397; 5,819,726;and 5,655,516, each of which is incorporated herein by reference. Theseapparatuses are particularly suitable as dry particle inhalers.

Aerosols of liquid particles comprising the active compound may beproduced by any suitable means, such as with a pressure-driven aerosolnebulizer (L C Star) or an ultrasonic nebulizer (Pari eFlow). See, e.g.,U.S. Pat. No. 4,501,729, which is incorporated herein by reference.Nebulizers are commercially available devices which transform solutionsor suspensions of the active ingredient into a therapeutic aerosol misteither by means of acceleration of compressed gas, typically air oroxygen, through a narrow venturi orifice or by means of ultrasonicagitation. Suitable formulations for use in nebulizers consist of theactive ingredient in a liquid carrier, the active ingredient comprisingup to 40% w/w of the formulation, but preferably less than 20% w/w. Thecarrier is typically water (and most preferably sterile, pyrogen-freewater) or dilute aqueous alcoholic solution. Perfluorocarbon carriersmay also be used. Optional additives include preservatives if theformulation is not made sterile, for example, methyl hydroxybenzoate,antioxidants, flavoring agents, volatile oils, buffering agents andsurfactants.

Aerosols of solid particles comprising the active compound may likewisebe produced with any solid particulate medicament aerosol generator.Aerosol generators for administering solid particulate medicaments to asubject produce particles which are respirable, as explained above, andgenerate a volume of aerosol containing predetermined metered dose ofmedicament at a rate suitable for human administration. One illustrativetype of solid particulate aerosol generator is an insufflator. Suitableformulations for administration by insufflation include finelycomminuted powders which may be delivered by means of an insufflator ortaken into the nasal cavity in the manner of a snuff. In theinsufflator, the powder (e.g., a metered dose thereof effective to carryout the treatments described herein) is contained in capsules orcartridges, typically made of gelatin or plastic, which are eitherpierced or opened in situ and the powder delivered by air drawn throughthe device upon inhalation or by means of a manually-operated pump. Thepowder employed in the insufflator consists either solely of the activeingredient or of powder blend comprising the active ingredient, asuitable powder diluent, such as lactose, and an optional surfactant.The active ingredient typically comprises of 0.1 to 100% w/w of theformulation. A second type of illustrative aerosol generator comprises ametered dose inhaler. Metered dose inhalers are pressurized aerosoldispensers, typically containing a suspension or solution formulation ofactive ingredient in a liquified propellant. During use, these devicesdischarge the formulation through a valve adapted to deliver a meteredvolume, typically from 10 to 150 μl, to produce a fine particle spraycontaining the active ingredient. Suitable propellants include certainchlorofluorocarbon compounds, for example, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof.The formulation may additionally contain one of more co-solvents, forexample, ethanol, surfactants, such as oleic acid or sorbitan trioleate,antioxidants and suitable flavoring agents.

The aerosol, whether formed from solid or liquid particles, may beproduced by the aerosol generator at a rate of from about 10 to 150liters per minute, more preferable from 30 to 150 liters per minute, andmost preferably about 60 liters per minute. Aerosols containing greateramounts of medicament may be administered more rapidly.

The dosage of the active compounds disclosed herein will vary dependingon the condition being treated and the state of the subject, butgenerally may be from about 0.01, 0.03, 0.05, 0.1 to 1, 5, 10 or 20 mgof the pharmaceutic agent, deposited on the airway surfaces. The dailydose may be divided among one or multiple unit dose administrations. Thegoal is to achieve a concentration of the pharmaceutic agents on lungairway surfaces of between 10⁻⁹-10⁴ M.

In another embodiment, they are administered by administering an aerosolsuspension of respirable or non-respirable particles (preferablynon-respirable particles) comprised of active compound, which thesubject inhales through the nose. The respirable or non-respirableparticles may be liquid or solid. The quantity of active agent includedmay be an amount of sufficient to achieve dissolved concentrations ofactive agent on the airway surfaces of the subject of from about 10⁻⁹,10⁻⁸, or 10⁻⁷ to about 10⁻³, 10⁻², 10⁻¹ moles/liter, and more preferablyfrom about 10⁻⁹ to about 10⁻⁴ moles/liter.

The dosage of active compound will vary depending on the condition beingtreated and the state of the subject, but generally may be an amountsufficient to achieve dissolved concentrations of active compound on thenasal airway surfaces of the subject from about 10⁻⁹, 10⁻⁸, 10⁻⁷ toabout 10⁻³, 10⁻², or 10⁻¹ moles/liter, and more preferably from about10⁻⁷ to about 10⁴ moles/liter. Depending upon the solubility of theparticular formulation of active compound administered, the daily dosemay be divided among one or several unit dose administrations. The dailydose by weight may range from about 0.01, 0.03, 0.1, 0.5 or 1.0 to 10 or20 milligrams of active agent particles for a human subject, dependingupon the age and condition of the subject. A currently preferred unitdose is about 0.5 milligrams of active agent given at a regimen of 2-10administrations per day. The dosage may be provided as a prepackagedunit by any suitable means (e.g., encapsulating a gelatin capsule).

In one embodiment of the invention, the particulate active agentcomposition may contain both a free base of active agent and apharmaceutically acceptable salt to provide both early release andsustained release of active agent for dissolution into the mucussecretions of the nose. Such a composition serves to provide both earlyrelief to the patient, and sustained relief over time. Sustained relief,by decreasing the number of daily administrations required, is expectedto increase patient compliance with the course of active agenttreatments.

Pharmaceutical formulations suitable for airway administration includeformulations of solutions, emulsions, suspensions and extracts. Seegenerally, J. Nairn, Solutions, Emulsions, Suspensions and Extracts, inRemington: The Science and Practice of Pharmacy, chap. 86 (19^(th) ed.1995), incorporated herein by reference. Pharmaceutical formulationssuitable for nasal administration may be prepared as described in U.S.Pat. No. 4,389,393 to Schor; U.S. Pat. No. 5,707,644 to Ilium; U.S. Pat.No. 4,294,829 to Suzuki; and U.S. Pat. No. 4,835,142 to Suzuki, thedisclosures of which are incorporated by reference herein in theirentirety.

Mists or aerosols of liquid particles comprising the active compound maybe produced by any suitable means, such as by a simple nasal spray withthe active agent in an aqueous pharmaceutically acceptable carrier, suchas a sterile saline solution or sterile water. Administration may bewith a pressure-driven aerosol nebulizer or an ultrasonic nebulizer. Seee.g. U.S. Pat. Nos. 4,501,729 and 5,656,256, both of which areincorporated herein by reference. Suitable formulations for use in anasal droplet or spray bottle or in nebulizers consist of the activeingredient in a liquid carrier, the active ingredient comprising up to40% w/w of the formulation, but preferably less than 20% w/w. Typicallythe carrier is water (and most preferably sterile, pyrogen-free water)or dilute aqueous alcoholic solution, preferably made in a 0.12% to 0.8%solution of sodium chloride. Optional additives include preservatives ifthe formulation is not made sterile, for example, methylhydroxybenzoate, antioxidants, flavoring agents, volatile oils,buffering agents, osmotically active agents (e.g. mannitol, xylitol,erythritol) and surfactants.

Compositions containing respirable or non-respirable dry particles ofmicronized active agent may be prepared by grinding the dry active agentwith a mortar and pestle, and then passing the micronized compositionthrough a 400 mesh screen to break up or separate out largeagglomerates.

The particulate composition may optionally contain a dispersant whichserves to facilitate the formation of an aerosol. A suitable dispersantis lactose, which may be blended with the active agent in any suitableratio (e.g., a 1 to 1 ratio by weight).

The compound of formula I may be synthesized according to proceduresknown in the art. A representative synthetic procedure is shown in thescheme below:

These procedures are described in, for example, E. J. Cragoe, “TheSynthesis of Amiloride and Its Analogs” (Chapter 3) in Amiloride and ItsAnalogs, pp. 25-36, incorporated herein by reference. Other methods ofpreparing the compounds are described in, for example, U.S. Pat. No.3,313,813, incorporated herein by reference. See in particular MethodsA, B, C, and D described in U.S. Pat. No. 3,313,813. Additional methodsof preparing intermediates used in the preparation of compounds of theinstant invention are disclosed in U.S. Pat. No. 7,064,129, U.S. Pat.No. 6,858,615, U.S. Pat. No. 6,903,105, WO 2004/073629, WO 2007/146869,and WO 2007/018640, each of which is expressly incorporated byreference.

Several assays may be used to characterize the compounds of the presentinvention. Representative assays are discussed below.

In Vitro Measure of Sodium Channel Blocking Activity and Reversibility

One assay used to assess mechanism of action and/or potency of thecompounds of the present invention involves the determination of lumenaldrug inhibition of airway epithelial sodium currents measured undershort circuit current (I_(SC)) using airway epithelial monolayersmounted in Ussing chambers. Cells obtained from freshly excised human,dog, sheep or rodent airways are seeded onto porous 0.4 micron Snapwell™Inserts (CoStar), cultured at air-liquid interface (ALI) conditions inhormonally defined media, and assayed for sodium transport activity(I_(SC)) while bathed in Krebs Bicarbonate Ringer (KBR) in Usingchambers. All test drug additions are to the lumenal bath with half-logdose addition protocols (from 1×10⁻¹¹ M to 3×10⁻⁵ M), and the cumulativechange in I_(sc) (inhibition) recorded. All drugs are prepared indimethyl sulfoxide as stock solutions at a concentration of 1×10⁻² M andstored at −20° C. Eight preparations are typically run in parallel; twopreparations per run incorporate amiloride and/or benzamil as positivecontrols. After the maximal concentration (5×10⁻⁵ M) is administered,the lumenal bath is exchanged three times with fresh drug-free KBRsolution, and the resultant I_(SC) measured after each wash forapproximately 5 minutes in duration. Reversibility is defined as thepercent return to the baseline value for sodium current after the thirdwash. All data from the voltage clamps are collected via a computerinterface and analyzed off-line.

Dose-effect relationships for all compounds are considered and analyzedby the Prism 3.0 program. IC₅₀ values, maximal effective concentrations,and reversibility are calculated and compared to amiloride and benzamilas positive controls. The potency of the sodium channel blockingactivity of representative compounds in freshly excised cell from canineairways is shown in Table 2.

TABLE 2 In Vitro Measure of Sodium Channel Blocking Activity CompoundIC₅₀ (nM) I 5.3 amiloride 781Pharmacological Assays of Absorption(1) Apical Disappearance Assay

Bronchial cells (dog, human, sheep, or rodent cells) are seeded at adensity of 0.25×10⁶/cm² on a porous Transwell-Col collagen-coatedmembrane with a growth area of 1.13 cm² grown at an air-liquid interfacein hormonally defined media that promotes a polarized epithelium. From12 to 20 days after development of an air-liquid interface (ALI) thecultures are expected to be >90% ciliated, and mucins will accumulate onthe cells. To ensure the integrity of primary airway epithelial cellpreparations, the transepithelial resistance (R_(t)) and transepithelialpotential differences (PD), which are indicators of the integrity ofpolarized nature of the culture, are measured. Human cell systems arepreferred for studies of rates of absorption from apical surfaces. Thedisappearance assay is conducted under conditions that mimic the “thin”films in vivo (˜25 μl) and is initiated by adding experimental sodiumchannel blockers or positive controls (amiloride, benzamil, phenamil) tothe apical surface at an initial concentration of 10 μM. A series ofsamples (5 μl volume per sample) is collected at various time points,including 0, 5, 20, 40, 90 and 240 minutes. Concentrations aredetermined by measuring intrinsic fluorescence of each sodium channelblocker using a Fluorocount Microplate Flourometer or HPLC. Quantitativeanalysis employs a standard curve generated from authentic referencestandard materials of known concentration and purity. Data analysis ofthe rate of disappearance is performed using nonlinear regression, onephase exponential decay (Prism V 3.0).

2. Confocal Microscopy Assay of Amiloride Congener Uptake

Virtually all amiloride-like molecules fluoresce in the ultravioletrange. This property of these molecules may be used to directly measurecellular update using x-z confocal microscopy. Equimolar concentrationsof experimental compounds and positive controls including amiloride andcompounds that demonstrate rapid uptake into the cellular compartment(benzamil and phenamil) are placed on the apical surface of airwaycultures on the stage of the confocal microscope. Serial x-z images areobtained with time and the magnitude of fluorescence accumulating in thecellular compartment is quantitated and plotted as a change influorescence versus time.

3. In Vitro Assays of Compound Metabolism

Airway epithelial cells have the capacity to metabolize drugs during theprocess of transepithelial absorption. Further, although less likely, itis possible that drugs can be metabolized on airway epithelial surfacesby specific ectoenzyme activities. Perhaps more likely as anecto-surface event, compounds may be metabolized by the infectedsecretions that occupy the airway lumens of patients with lung disease,e.g. cystic fibrosis. Thus, a series of assays is performed tocharacterize the compound metabolism that results from the interactionof test compounds with human airway epithelia and/or human airwayepithelial lumenal products.

In the first series of assays, the interaction of test compounds in KBRas an “ASL” stimulant are applied to the apical surface of human airwayepithelial cells grown in the T-Col insert system. For most compounds,metabolism (generation of new species) is tested for using highperformance liquid chromatography (HPLC) to resolve chemical species andthe endogenous fluorescence properties of these compounds to estimatethe relative quantities of test compound and novel metabolites. For atypical assay, a test solution (25 μl KBR, containing 10 μM testcompound) is placed on the epithelial lumenal surface. Sequential 5 to10 μl samples are obtained from the lumenal and serosal compartments forHPLC analysis of (1) the mass of test compound permeating from thelumenal to serosal bath and (2) the potential formation of metabolitesfrom the parent compound. In instances where the fluorescence propertiesof the test molecule are not adequate for such characterizations,radiolabeled compounds are used for these assays. From the HPLC data,the rate of disappearance and/or formation of novel metabolite compoundson the lumenal surface and the appearance of test compound and/or novelmetabolite in the basolateral solution is quantitated. The data relatingthe chromatographic mobility of potential novel metabolites withreference to the parent compound are also quantitated.

To analyze the potential metabolism of test compounds by CF sputum, a“representative” mixture of expectorated CF sputum obtained from 10 CFpatients (under IRB approval) has been collected. The sputum has been besolubilized in a 1:5 mixture of KBR solution with vigorous vortexing,following which the mixture was split into a “neat” sputum aliquot andan aliquot subjected to ultracentrifugation so that a “supernatant”aliquot was obtained (neat=cellular; supernatant=liquid phase). Typicalstudies of compound metabolism by CF sputum involve the addition ofknown masses of test compound to “neat” CF sputum and aliquots of CFsputum “supernatant” incubated at 37° C., followed by sequentialsampling of aliquots from each sputum type for characterization ofcompound stability/metabolism by HPLC analysis as described above. Asabove, analysis of compound disappearance, rates of formation of novelmetabolities, and HPLC mobilities of novel metabolites are thenperformed.

4. Pharmacological Effects and Mechanism of Action of the Drug inAnimals

The effect of compounds for enhancing mucociliary clearance (MCC) can bemeasured using an in vivo model described by Sabater et al., Journal ofApplied Physiology, 1999, pp. 2191-2196, incorporated herein byreference.

Methods

Animal Preparation:

Adult ewes (ranging in weight from 25 to 35 kg) were restrained in anupright position in a specialized body harness adapted to a modifiedshopping cart. The animals' heads were immobilized and local anesthesiaof the nasal passage was induced with 2% lidocaine. The animals werethen nasally intubated with a 7.5 mm internal diameter endotracheal tube(ETT). The cuff of the ETT was placed just below the vocal cords and itsposition was verified with a flexible bronchoscope. After intubation theanimals were allowed to equilibrate for approximately 20 minutes priorto initiating measurements of mucociliary clearance.

Administration of Radio-Aerosol:

Aerosols of ^(99m)Tc-Human serum albumin (3.1 mg/ml; containingapproximately 20 mCi) were generated using a Raindrop Nebulizer whichproduces a droplet with a median aerodynamic diameter of 3.6 μm. Thenebulizer was connected to a dosimetry system consisting of a solenoidvalve and a source of compressed air (20 psi). The output of thenebulizer was directed into a plastic T connector; one end of which wasconnected to the endotracheal tube, the other was connected to a pistonrespirator. The system was activated for one second at the onset of therespirator's inspiratory cycle. The respirator was set at a tidal volumeof 500 mL, an inspiratory to expiratory ratio of 1:1, and at a rate of20 breaths per minute to maximize the central airway deposition. Thesheep breathed the radio-labeled aerosol for 5 minutes. A gamma camerawas used to measure the clearance of ^(99m)Tc-Human serum albumin fromthe airways. The camera was positioned above the animal's back with thesheep in a natural upright position supported in a cart so that thefield of image was perpendicular to the animal's spinal cord. Externalradio-labeled markers were placed on the sheep to ensure properalignment under the gamma camera. All images were stored in a computerintegrated with the gamma camera. A region of interest was traced overthe image corresponding to the right lung of the sheep and the countswere recorded. The counts were corrected for decay and expressed aspercentage of radioactivity present in the initial baseline image. Theleft lung was excluded from the analysis because its outlines aresuperimposed over the stomach and counts can be swallowed and enter thestomach as radio-labeled mucus.

Treatment Protocol (Assessment of Activity at t-Zero):

A baseline deposition image was obtained immediately after radio-aerosoladministration. At time zero, after acquisition of the baseline image,vehicle control (distilled water), positive control (amiloride), orexperimental compounds were aerosolized from a 4 ml volume using a PariLC JetPlus nebulizer to free-breathing animals. The nebulizer was drivenby compressed air with a flow of 8 liters per minute. The time todeliver the solution was 10 to 12 minutes. Animals were extubatedimmediately following delivery of the total dose in order to preventfalse elevations in counts caused by aspiration of excess radio-tracerfrom the ETT. Serial images of the lung were obtained at 15-minuteintervals during the first 2 hours after dosing and hourly for the next6 hours after dosing for a total observation period of 8 hours. Awashout period of at least 7 days separated dosing sessions withdifferent experimental agents.

Treatment Protocol (Assessment of Activity at t-4 Hours):

The following variation of the standard protocol was used to assess thedurability of response following a single exposure to vehicle control(distilled water), positive control compounds (amiloride or benzamil),or investigational agents. At time zero, vehicle control (distilledwater), positive control (amiloride), or investigational compounds wereaerosolized from a 4 ml volume using a Pari LC JetPlus nebulizer tofree-breathing animals. The nebulizer was driven by compressed air witha flow of 8 liters per minute. The time to deliver the solution was 10to 12 minutes. Animals were restrained in an upright position in aspecialized body harness for 4 hours. At the end of the 4-hour periodanimals received a single dose of aerosolized ^(99m)Tc-Human serumalbumin (3.1 mg/ml; containing approximately 20 mCi) from a RaindropNebulizer. Animals were extubated immediately following delivery of thetotal dose of radio-tracer. A baseline deposition image was obtainedimmediately after radio-aerosol administration. Serial images of thelung were obtained at 15-minute intervals during the first 2 hours afteradministration of the radio-tracer (representing hours 4 through 6 afterdrug administration) and hourly for the next 2 hours after dosing for atotal observation period of 4 hours. A washout period of at least 7 daysseparated dosing sessions with different experimental agents.

Statistics: Data were analyzed using SYSTAT for Windows, version 5. Datawere analyzed using a two-way repeated ANOVA (to assess overalleffects), followed by a paried t-test to identify differences betweenspecific pairs. Significance was accepted when P was less than or equalto 0.05. Slope values (calculated from data collected during the initial45 minutes after dosing in the t-zero assessment) for mean MCC curveswere calculated using linear least square regression to assessdifferences in the initial rates during the rapid clearance phase.

EXAMPLES

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

Preparation of Sodium Channel Blockers

Materials and methods. All reagents and solvents were purchased fromAldrich Chemical Corp. and used without further purification. Proton andcarbon NMR spectra were obtained on a Bruker AC 300 spectrometer at 300MHz and 75 MHz, respectively. Proton spectra were referenced totetramethylsilane as an internal standard and the carbon spectra werereferenced to CDCl₃, CD₃OD, acetone-d₆ or DMSO-d₆ (purchased fromAldrich or Cambridge Isotope Laboratories, unless otherwise specified).Melting points were obtained on a Mel-Temp II apparatus and areuncorrected. ESI Mass spectra were obtained on a Shimadzu LCMS-2010 EVMass Spectrometer. HLPC analyses were obtained using a Waters XTerra RPC18 Analytical Column detected at 220 nm (unless otherwise specified) ona Shimadzu Prominence HPLC system. With a flow rate of 1.0 mL perminute, the following time program was utilized:

Percent A Percent B Time (H₂O with 0.05% TFA) (CH₃CN with 0.05% TFA) 0:00 90 10 20:00 10 90 30:00 10 90 35:00 90 10

The following definitions for abbreviations will apply unless otherwiseindicated.

Abbreviation Definition THF tetrahydrofuran Cbz Benzyloxycarbonyl i.e.—(CO)O-benzyl AUC Area under the curve or peak EtOAc Ethyl acetate R_(f)Retardation factor HPLC High performance liquid chromatography MTBEMethyl tertiary butyl ether t_(R) Retention time GC-MS Gaschromatography-mass spectrometry wt % Percent by weight h hours minminutes MHz megahertz MeOH methanol TFA Trifluoroacetic acid UVUltraviolet

Preparation of Formula I (15)

Preparation of Compound 2

Acetyl chloride (27.1 mL, 380.1 mmol) was added drop wise to MeOH (225mL) at room temperature, and compound 1 (15.0 g, 54.3 mmol) was added.The resulting solution was refluxed overnight and concentrated, thencrashed from hexanes (700 mL) to afford desired compound 2 (14.0 g, 89%)as an off-white solid: ¹H NMR (300 MHz, CD₃OD) δ 7.07 (d, J=8.7 Hz, 2H),6.71 (d, J=8.7 Hz, 2H), 4.02 (t, J=6.6 Hz, 1H), 3.68 (s, 3H), 2.75-2.57(m, 2H), 2.22-2.05 (m, 2H).

Preparation of Compound 3

To a solution of compound 2 (14.0 g, 48.2 mmol) in MeOH/H₂O (140 mL/70mL) was added NaHCO₃ (28.5 g, 337.4 mmol) and Boc₂O (11.5 g, 53.0 mmol)at 0° C. The resulting mixture was allowed to warm to room temperatureand stirred overnight. The reaction mixture was partitioned betweenEtOAc (300 mL) and water (300 mL) The aqueous layer was separated andextracted with EtOAc (2×300 mL). The combined organic extracts werewashed with brine, dried over Na₂SO₄ and concentrated to give compound 3(13.5 g, 90%) as an off-white solid: ¹H NMR (300 MHz, CD₃OD) δ 6.98 (d,J=8.4 Hz, 2H), 6.67 (d, J=8.4 Hz, 2H), 4.03 (t, J=4.5 Hz, 1H), 3.68 (s,3H), 2.65-2.40 (m, 2H), 1.92-1.82 (m, 1H), 1.44 (s, 9H).

Preparation of Compound 4

To a solution of compound 3 (13.5 g, 43.6 mmol) in pyridine (150 mL) wasadded triflate (7.3 mL, 43.6 mmol) at 0° C., and the reaction mixturewas stirred at 0° C. for 3 h. After concentrated, the reaction mixturewas partitioned between EtOAc (300 mL) and water (300 mL). The aqueouslayer was separated and extracted with EtOAc (2×300 mL). The combinedorganic extracts were washed with brine, dried over Na₂SO₄ andconcentrated. The residue was purified by column chromatography (silicagel, 95:5 CHCl₃/MeOH) to afford desired compound 4 (16.0 g, 83%) as anoff brown solid: ¹H NMR (300 MHz, CDCl₃) δ 7.26-7.23 (m, 2H), 7.20-7.17(m, 2H), 5.08 (br s, 1H), 4.36 (br s, 1H), 3.71 (s, 3H), 2.73-2.67 (m,2H), 2.21-2.13 (m, 1H), 1.99-1.87 (m, 1H), 1.45 (s, 9H).

Preparation of Compound 6

To a solution of compound 4 (16.0 g, 36.2 mmol) in anhydrous CH₃CN (160mL) was added TEA (20.9 mL, 144.8 mmol), 10% (t-Bu)₃P in hexanes (1.4 g,7.2 mmol), benzyl but-3-ynylcarbamate (5, 8.8 g, 43.4 mmol) and CuI (340mg, 1.8 mmol) at room temperature. The resulting mixture was degassedwith Argon for 3 min and Pd(PPh₃)₄ (4.1 g, 3.6 mmol) was added rapidlyin one portion. After degassed with Argon for 5 min, the resultingmixture was heated at 60° C. for 12 h. The reaction mixture wasconcentrated in vacuum and the residue was purified by column (silicagel, 40:60 hexanes/EA) to afford compound 6 (16.0 g, 70%) as a brownoil: ¹H NMR (300 MHz, CDCl₃) δ 7.36-7.28 (m, 8H), 7.07 (d, J=8.4 Hz,2H), 5.12 (br s, 4H), 4.33 (br s, 1H), 3.71 (s, 3H), 3.46-3.39 (m, 2H),2.68-2.59 (m, 4H), 2.14-2.20 (m, 1H), 1.96-1.84 (m, 1H), 1.44 (s, 9H).

Preparation of Compound 7

Compound 6 (8.0 g, 16.1 mmol) was dissolved in 4 N HCl in dioxane (60mL) at room temperature and the resulting solution was stirred for 1 h.After concentrated, the residue was crashed from MTBE to affordhydrochloric acid salt 7 (6.75 g, 90%) as a white solid: ¹H NMR (300MHz, CD₃OD) δ 7.32-7.28 (m, 7H), 7.18 (d, J=8.1 Hz, 2H), 5.08 (s, 2H),4.04 (t, J=6.3 Hz, 1H), 3.82 (s, 3H), 3.31-3.29 (m, 2H), 2.82-2.67 (m,2H), 2.58 (t, J=6.9 Hz, 2H), 2.26-2.08 (m, 2H).

Preparation of Compound 8

To a solution of compound 7 (6.75 g, 15.6 mmol) in MeOH/AcOH (70 mL/15mL) was added 37% CH₂O in H₂O (25.2 mL, 312 mmol) and sodiumcyanoborohydride (7.84 g, 124.8 mmol). The reaction mixture was stirredat room temperature overnight. After concentrated, the residue waspurified by column chromatography (silica gel, 10:1 CH₂Cl₂/MeOH,10:1:0.1 CHCl₃/MeOH/NH₄OH) to afford desired compound 8 (3.8 g, 63%) asa off-white solid: NMR (300 MHz, CDCl₃) δ 7.36-7.26 (m, 7H), 7.11 (d,J=8.1 Hz, 2H), 5.11 (s, 2H), 3.69 (s, 3H), 3.45-3.39 (m, 2H), 3.10 (t,J=7.5 Hz, 1H), 2.65-2.60 (m, 4H), 2.32 (s, 6H), 2.01-1.88 (m, 2H).

Preparation of Compound 9

To a solution of methyl ester 8 (3.80 g, 8.9 mmol) in THF/MeOH/H₂O (30mL/30 mL/10 mL) was added NaOH (3.50 g, 89.0 mmol). The reaction mixturewas stirred at room temperature overnight. After concentrated, theresidue was dissolved in H₂O (50 mL) and 1 N aq HCl was added to adjustpH value to 5. The resulting precipitate was filtered out and dried toafford desired compound 9 (3.25 g, 90%) as an off-white solid: NMR (300MHz, CD₃OD) δ 7.32-7.28 (m, 7H), 7.17 (d, J=8.4 Hz, 2H), 5.08 (s, 2H),3.46-3.42 (m, 1H), 3.45-3.39 (m, 2H), 2.83-2.70 (m, 8H), 2.58 (t, J=6.9Hz, 2H), 2.15-2.05 (m, 2H).

Preparation of Compound 11

To a solution of compound 9 (1.80 g, 4.4 mmol) in anhydrous DMF (20 mL)was added HATU (3.3 g, 8.8 mmol) and DIPEA (4.0 mL, 22.0 mmol). Theresulting solution was stirred at room temperature for 0.5 h and amine10 (2.1 g, 5.2 mmol) was added. The reaction mixture was stirred at roomtemperature overnight and partitioned between EtOAc (200 mL) and water(200 mL). The aqueous layer was separated and extracted with EtOAc(2×200 mL). The combined organic extracts were washed with brine, driedover Na₂SO₄ and concentrated. The residue was purified by columnchromatography (silica gel, 95:5 CHCl₃/MeOH) to afford desired compound11 (1.70 g, 49%) as a white solid: ¹H NMR (300 MHz, CDCl₃) δ 7.36-7.28(m, 7H), 7.10 (d, J=8.1 Hz, 2H), 5.10 (s, 3H), 4.97-4.90 (m, 1H), 3.86(t, J=6.6 Hz, 2H), 3.45-3.35 (m, 2H), 3.12 (s, 3H), 3.09-3.00 (m, 2H),2.96 (s, 3H), 2.91-2.71 (m, 2H), 2.62 (t, J=6.3 Hz, 2H), 2.30 (s, 6H),1.97-1.31 (m, 26H).

Preparation of Compound 12

A suspension of compound 11 (1.7 g, 2.1 mmol) and 10% Pd/C (800 mg) inMeOH/AcOH (25 mL/1.0 mL) was subjected to hydrogenation conditions (1atm) overnight at room temperature. The reaction mixture was filteredthrough celite and washed with MeOH. The filtrate was concentrated invacuum and crashed from MTBE to afford acetic salt 12 (1.45 g, 90%) ascolorless oil: ¹H NMR (400 MHz, CD₃OD) δ 7.12 (s, 4H), 4.97-4.90 (m,2H), 3.90-3.85 (m, 2H), 3.29 (s, 3H), 2.95 (s, 3H), 2.91 (t, J=7.6 Hz,2H), 2.63 (t, J=6.8 Hz, 2H), 2.57-2.50 (m, 8H), 1.95 (s, 6H), 2.02-1.96(m, 2H), 1.80-1.62 (m, 10H), 1.50 (s, 9H), 1.45 (s, 9H).

Preparation of Compound 14

To a solution of acetic acid salt 12 (1.45 g, 2.1 mmol) and methyl3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate hydroiodicacid salt (13, 1.28 g, 3.3 mmol) in EtOH (20 mL) was added DIPEA (2.17g, 16.8 mmol) at room temperature. The reaction mixture was heated at70° C. for 2 h, then cooled to room temperature and concentrated invacuum. The residue was purified by column chromatography (silica gel,9:1 CH₂Cl₂/MeOH, 80:18:2 CHCl₃/MeOH/NH₄OH) to afford desired compound 14(1.15 g, 68%) as a yellow solid: ¹H NMR (400 MHz, CD₃OD) δ 7.09 (s, 4H),4.87-4.82 (m, 2H), 3.86 (t, J=7.2 Hz, 2H), 3.24-3.18 (m, 3H), 3.22 (s,3H), 2.96-2.91 (m, 4H), 2.64-2.47 (m, 4H), 2.30 (t, 6H), 1.99-1.72 (m,10H), 1.55-1.32 (m, 20H).

Preparation of Compound 15 [FormulaI]-3,5-diamino-6-chloro-N—(N-(4-(4-((S)-3-(dimethylamino)-4-((S)-1-(dimethylamino)-6-guanidino-1-oxohexan-2-ylamino)-4-oxobutyl)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide

To a solution of compound 14 (670 mg, 0.7 mmol) in CH₂Cl₂ (20 mL) wasadded TFA (5.0 mL) and the resulting solution was stirred at roomtemperature for 2 h. The reaction mixture was concentrated in vacuum andazeotroped with 1 N aq HCl for 3 times to afford hydrochloric acid saltof 15 (Formula I, 600 mg, 99%) as a yellow hygroscopic solid: ¹H NMR(400 MHz, D₂O) δ 7.16-7.15 (m, 4H), 3.84-3.81 (m, 1H), 3.26 (br s, 2H),3.13-3.11 (m, 5H), 2.91-2.86 (m, 9H), 2.61-2.53 (m, 4H), 2.25-2.11 (m,2H), 1.74-1.23 (m, 10H).

Summary of in vitro data for Compound 15:

-   -   Potency (IC₅₀) on Canine Bronchial Epithelial Cells: 5.3±3.3 nM        (n=5) (FIG. 1)    -   Reversal of Maximal Effect on Canine Bronchial Epithelial Cells:        6.8±5.1% (n=3) of effect was lost with 3 washes    -   Absorption Rate Across Human Bronchial Epithelial Cells:        0.12±0.05 nM/cm²/min (n=3)    -   Metabolism by Human Bronchial Epithelial: No metabolites        detected apical or basolateral    -   Human Plasma Stability: No metabolism detected over 4 hours    -   Durability of Retention of Airway Surface Liquid by Canine        Bronchial Epithelial Cells: 94±5% (n=3) of fluid retained 8        hours after drug delivery; 56±6% (n=2) of fluid retained 24        hours after drug delivery    -   Rat Hepatocyte Metabolism: No evidence of significant metabolism        throughout 24-hour incubation    -   Human Plasma Protein Binding: 80±5% (n=6) bound to plasma        proteins

A Dose Response Curve of ENaC Inhibition with P-1046 (I) and Amilorideis shown in FIG. 1.

Ocular Data and Methods for Compound 15 (Formula I, aka I, aka P-1046,aka 1046):

The Effects of P-1046 (Formula I, 15) Concentration on Tear Output inExLac Rats is shown in FIG. 2

The Clearance of P-1046 (Formula I, 15) from the Ocular Surface is shownin FIG. 3.

The Effects of 10 mM P-1046 (Formula I, 15) on Tear Output in ExLac Ratsis shown in FIG. 4.

Procedures for Phenol Red Thread (PRT) Measurement of Tear Output:

Animal Species/Strain: Rats, Sprague Dawley (SD); Lacrimal glandsurgically removed (ExLac).

Animal Number/Sex: 3-4 females/group

Test Article Formulation: All stock solutions for each dosing arm wereprepared no more than 48 hours prior to the start of the study. Theconcentration of ENaC blockers in all test article solutions wereconfirmed by spectrophotometry.

Test Article Administration: Both the ipsilateral and contralateral eyeswere dosed with 5 □l of test article solution.

Phenol Red Thread (PRT) Test: Tear production was measured using theZoneQuick cotton thread with impregnated phenol red dye. The folded endof the thread was held in the lateral-ventral conjunctival cul-de-sacfor 10 seconds. The length of tear wicking onto the thread wasdetermined by measuring the length of the thread that changes color fromyellow to red. Use of a stereomicroscope was assist in the accuratemeasurement (recorded in millimeters) of the wicking/color change.Procedure for P-1046 (Compound 15 Formula I, aka I, aka P-1046, aka1046): Extraction from PRT Threads:All PRT threads were collected in eppendorf tubes and stored at −20 Cuntil the time of drug concentration analysis. P-1046 was extracted fromthreads and analyzed as follows:

-   -   1. 200 μL of 70% ACN (70% ACN, 30% H₂O) was added into sample        tubes which have threads in them.    -   2. Tubes from Step 1 were vortexed for 30 seconds and sonicated        for 1 minute to fully immerse the threads.    -   3. The sample solutions were incubated for 4 hours at room        temperature.    -   4. Samples were vortexed again for 30 seconds.    -   5. 75 μL of the each sample solution (from step 4) was removed        into 96-well UPLC plate with an additional 75 μL of mobile phase        A (5 mM ammonium formate, 0.1% formic acid in H₂O) added into        each sample well.    -   6. Analytical standards (10 mM, 1 mM, 100 □M, 10 □M, 1 □M, 100        nM, Buffer) were prepared identically to the thread solutions.    -   7. Drug concentrations from all samples were analyzed by UPLC.        Solubility and Stability Testing for Compound 15 (Formula I, aka        I, aka P-1046, aka 1046):        Procedure for Solubility and Stability Evaluation:    -   1. Prepare 2.8% NaCl and 25 mM citrate buffer, pH of the        solution 4.2. The osmolality of the solution is approximately        940 mOsM.    -   2. Add appropriate amount of buffer solution to a test compound        to make approximate concentration of 10 mg/mL.    -   3. Vortex the solution for 15 seconds, sonicate for 30 seconds,        again vortex for 60 seconds and visually observe the solution.    -   4. Calculate final concentration of the solution by a        spectrophotometer.        -   Remove a portion of solution and dilute it to 1:10 ratio.            Put this solution at 50° C. for the accelerated stability            study for 10 days.        -   Obtain stability data by HPLC analysis    -   5. Prepare 2.8% NaCl and 25 mM citrate buffer, pH of the        solution 4.2. The osmolality of the solution is approximately        940 mOsM.    -   6. Add appropriate amount of buffer solution to a test compound        to make approximate concentration of 10 mg/mL.    -   7. Vortex the solution for 15 seconds, sonicate for 30 seconds,        again vortex for 60 seconds and visually observe the solution.    -   8. Calculate final concentration of the solution by a        spectrophotometer.        -   Remove a portion of solution and dilute it to 1:10 ratio.            Put this solution at 50° C. for the accelerated stability            study for 10 days.        -   Obtain stability data by HPLC analysis.

FIG. 5 shows HPLC Analysis of P-1046 (Formula I, 15)solubility/stability samples at Day 1 and Day 10.

Summary Safety and Tolerability for Compound 15 (Formula I, aka I, akaP-1046, aka 1046):

An Acute Non-GLP Ocular Toxicity Study in New Zealand White Rabbits withP-1046

Objective:

The purpose of this study was to evaluate the ocular tolerability andsystemic exposure of P-1046, an inhibitor of the epithelial sodiumchannel (ENaC), when administered as a topical instillation to NewZealand white rabbits.

Methods:

The test article (P-1046) were supplied as a light yellow white powder.The test articles were then prepared into dosing solutions for topicalocular application. Twenty-eight experimentally naïve male New ZealandWhite rabbits, approximately 4 months old at the outset of the study andweighing 2.6-3.1 kilograms at randomization were assigned to treatmentgroups as shown in the table below:

P-1046 Concentration Number Number Group (Right Eye)* of doses** ofAnimals 1. Vehicle 0 8 4 6. P-1046 - low dose 10 8 4 7. P-1046 - highdose 30 4 4 *At each dose, 50 μl of vehicle or test article solution wasinstilled onto the right eye and 50 μl of saline was instilled onto theleft eye **Doses were administered eight times (1 hr between doses, 10mM dose groups) or four times (2 hr between each dose, 30 mM dosegroups) during a single day

Animals were administered 50 μl of the test or control vehicle onto theglobe of right eye and 50 μl of saline onto the globe of the left eyeeight times (approximately one hour between each dose) in Groups 1 and 6and four times (approximately two hours between each dose) in Group 7.Mortality and clinical observations were recorded daily. Body weightswere recorded at randomization/selection and prior to sacrifice on Day2. For blink rate assessment, eye blinks in the right eye only werecounted for 3 minutes prior to treatment initiation and following alldoses. Instances of wincing and pawing at the eyes during the 3-minuteeye blink observation period were recorded as clinical observations.Food consumption was recorded daily. Eyes were scored according toDraize prior to treatment, following each dose and on Day 2 atapproximately 24 hours post-dose. Blood for toxicokinetic evaluation wascollected from animals in Group 2-7 at pre-dose, 30 minutes after themiddle dose (4^(th) dose in groups 3, 5, and 6; 2^(nd) dose in groups 2,4, and 7) and at 30, 60, 120 and 240 minutes following the final dose onDay 1. Blood samples for evaluation of serum chemistry parameters werecollected prior to treatment initiation and at 120 and 240 min followingthe final dose. Urine samples were collected over a 4-hour periodfollowing the final dose. All animals were sacrificed followingcompletion of study procedures on Day 2.

Results and Conclusions:

Animals on study appeared to be in good overall health, as judged byclinical observations, body weights and food consumption.

Ocular irritation was assessed by blink rate measurement, Draizeevaluation of the eyes and clinical observations. Irritation associatedwith each treatment is discussed below.

Vehicle

The vehicle was well tolerated with only minor, transient ocularirritation following dosing. The vehicle control animals hadconjunctival redness in both eyes (max Draize score=1) and isolatedincidences of wincing and pawing at the eyes. The incidence ofconjunctival redness was slightly higher in eyes treated with vehicle(right eye), as compared to the eyes treated with saline (left eye),which suggests that observed irritation may have been the result of bothvehicle effects and the dosing procedure (FIG. 6). All eyes appearednormal on Day 2.

10 and 30 mM P-1046

The P-1046 formulations were well tolerated. Post-dose conjunctivalredness was observed, but the severity and incidence of this sign werecomparable to the vehicle controls. All eyes in the 10 mM group appearednormal on Day 2.

A Summary of P-1046 Ocular Tolerability is shown in FIG. 6.

FIG. 7 shows P-1046 Plasma Levels after Ocular Dosing.

What is claimed is:
 1. A compound represented by the formula (I):

and racemates, enantiomers, diastereomers, tautomers, polymorphs,pseudopolymorphs and pharmaceutically acceptable salts, thereof.
 2. Thecompound of claim 1, which is an acid addition salt of an inorganic acidor an organic acid selected from the group consisting of hydrochloricacid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid,acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid,furmaric acid, gluconic acid, citric acid, malic acid, ascorbic acid,benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamicacid, naphthalensulfonic acid, methanesulfonic acid, p-toluenesulfonicacid, naphthalenedisulfonic acid, polygalacturonic acid, malonic acid,sulfosalicylic acid, glycolic acid, 2-hydroxy-3-naphthoate, pamoate,salicylic acid, stearic acid, phthalic acid, mandelic acid, and lacticacid.
 3. A pharmaceutical composition, comprising the compound of claim1 and a pharmaceutically acceptable carrier.
 4. A composition,comprising: the compound of claim 1; and a P2Y2 receptor agonist.
 5. Acomposition, comprising: the compound of claim 1; and a bronchodilator.6. A method of promoting hydration of mucosal surfaces, comprising:administering an effective amount of the compound of claim 1 to amucosal surface of a subject.
 7. A method of blocking sodium channels,comprising: contacting sodium channels with an effective amount of thecompound of claim
 1. 8. A method of treating a disease ameliorated byincreased mucociliary clearance and mucosal hydration comprisingadministering to a subject in need of increased mucociliary clearanceand mucosal hydration an effective amount of an osmolyte and thecompound of claim
 1. 9. The method of claim 8, wherein the compound isadministered preceding administration of the osmolyte.
 10. The method ofclaim 8, wherein the compound is administered concurrent withadministration of the osmolyte.
 11. The method of claim 8, wherein thecompound is administered following administration of the osmolyte. 12.The method of claim 8, wherein the osmolyte is hypertonic saline ormannitol.
 13. The method of claim 8, wherein the osmolyte is sodiumchloride which is delivered as a micronized particle of respirable size.14. The method of claim 8, wherein the effective amount of an osmolyteand a sodium channel blocker is administered by aerosolization using adevice capable of delivering the formulation to the nasal passages orpulmonary airway wherein the aerosol is a respirable size.
 15. Acomposition, comprising: (a) the compound of claim 1 and (b) anosmotically active compound.
 16. A method of inducing sputum, comprisingadministering to a subject in need of increased mucociliary clearanceand mucosal hydration an effective amount of an osmolyte and thecompound of claim 1.